A Linkage-specific Sialic Acid Labeling Strategy Reveals Different Site-specific Glycosylation Patterns in SARS-CoV-2 Spike Protein Produced in CHO and HEK Cell Substrates.

Qiong Wang,Yuan Tian,Michael J Betenbaugh,Hongpeng Jia,John F Cipollo,Yiqun Chen,Andrew Pekosz,Yan Wang,Lisa Parsons,Changyi Lin,Lateef Aliyu,Shuang Yang

doi:10.3389/fchem.2021.735558

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus utilizes the extensively glycosylated spike (S) protein protruding from the viral envelope to bind to angiotensin-converting enzyme-related carboxypeptidase (ACE2) as its primary receptor to mediate host-cell entry. Currently, the main recombinant S protein production hosts are Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells. In this study, a recombinant S protein truncated at the transmembrane domain and engineered to express a C-terminal trimerization motif was transiently produced in CHO and HEK cell suspensions. To further evaluate the sialic acid linkages presenting on S protein, a two-step amidation process, employing dimethylamine and ammonium hydroxide reactions in a solid support system, was developed to differentially modify the sialic acid linkages on the glycans and glycopeptides from the S protein. The process also adds a charge to Asp and Glu which aids in ionization. We used MALDI-TOF and LC-MS/MS with electron-transfer/higher-energy collision dissociation (EThcD) fragmentation to determine global and site-specific N-linked glycosylation patterns. We identified 21 and 19 out of the 22 predicted N-glycosites of the SARS-CoV-2 S proteins produced in CHO and HEK, respectively. It was found that the N-glycosite at 1,158 position (N1158) and at 122, 282 and 1,158 positions (N122, N282 and N1158) were absent on S from CHO and HEK cells, respectively. The structural mapping of glycans of recombinant human S proteins reveals that CHO-Spike exhibits more complex and higher sialylation (α2,3-linked) content while HEK-Spike exhibits more high-mannose and a small amount of α2,3- and α2,6-linked sialic acids. The N74 site represents the most abundant glycosite on both spike proteins. The relatively higher amount of high-mannose abundant sites (N17, N234, N343, N616, N709, N717, N801, and N1134) on HEK-Spike suggests that glycan-shielding may differ among the two constructs. HEK-Spike can also provide different host immune system interaction profiles based on known immune system active lectins. Collectively, these data underscore the importance of characterizing the site-specific glycosylation of recombinant human spike proteins from HEK and CHO cells in order to better understand the impact of the production host on this complex and important protein used in research, diagnostics and vaccines.

Highlights

The ongoing outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a major global pandemic affecting the lives of billions of people worldwide (Yuen et al, 2020)
A research group discovered that SARS-COV-2 spike glycoprotein glycans shield approximately 40% of the protein surface, the glycans only account for 17% of the total molecular weight of the S trimer when expressed from human embryonic kidney cells (Grant et al, 2020; Yang et al, 2020)
Structural mapping of glycans of recombinant full-length human S proteins revealed that Chinese hamster ovary (CHO)-Spike presented more complex and higher sialylation (α2,3-linked only; ∼40% of total) content while HEKSpike had marginally more high-mannose glycan, almost exclusively as Man5GlcNAc2, and minor amounts of α2,3- and α2,6 linked sialylation

Summary

Introduction

The ongoing outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a major global pandemic affecting the lives of billions of people worldwide (Yuen et al, 2020). The glycan profile of envelope glycoproteins can affect the pathobiological activities and characteristics of many viruses, such as glycoprotein folding, trafficking and stability, virus release, functional activity, and immune evasion including shielding of the peptide backbone by glycans (Watanabe et al, 2019). Similar to the SARS outbreak in 2003, the SARS-CoV-2 spike utilizes glycan shielding to thwart the host immune response. A research group discovered that SARS-COV-2 spike glycoprotein glycans shield approximately 40% of the protein surface, the glycans only account for 17% of the total molecular weight of the S trimer when expressed from human embryonic kidney cells (Grant et al, 2020; Yang et al, 2020). Understanding the glycosylation of recombinant S protein is very important for studying virus biology and immune response, and help to provide information for the application of recombinant spike glycoprotein in diagnosis and vaccines

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Conclusion